Structure-Dynamic Coupling Through Ca²⁺-Binding Regulatory Domains of Mammalian NCX Isoform/Splice Variants

Mammalian Na⁺/Ca²⁺ exchangers (NCX1, NCX2, and NCX3) and their splice variants are expressed in a tissue-specific manner and are regulated by Ca²⁺ binding CBD1 and CBD2 domains. NCX2 does not undergo splicing, whereas in NCX1 and NCX3, the splicing segment (with mutually exclusive and cassette exons) is located in CBD2. Ca²⁺ binding to CBD1 results in Ca²⁺-dependent tethering of CBDs through the network of interdomain salt-bridges, which is associated with NCX activation, whereas a slow dissociation of "occluded" Ca²⁺ inactivates NCX. Although NCX variants share a common structural basis for Ca²⁺-dependent tethering of CBDs, the Ca²⁺ off-rates of occluded Ca²⁺ vary up to 50-fold, depending on the exons assembly. The Ca²⁺-dependent tethering of CBDs rigidifies the interdomain movements of CBDs without any significant changes in the CBDs' alignment; consequently, more constraining conformational states become more populated in the absence of global conformational changes. Although this Ca²⁺-dependent "population shift" is a common mechanism among NCX variants, the strength and span of backbone rigidification from the C-terminal of CBD1 to the C-terminal of CBD2 is exon dependent. The mutually exclusive exons differentially stabilize/destabilize the backbone dynamics of Ca²⁺-bound CBDs in NCX1 and NCX3 variants, whereas the cassette exons control the stability of the interdomain linker. The combined effects of mutually exclusive and cassette exons permit a fine adjustment of two different regulatory pathways: the Ca²⁺-dependent activation (controlled by CBD1) and the Ca²⁺-dependent alleviation of Na⁺-induced inactivation (controlled by CBD2). Exon-controlled dynamic features match with cell-specific regulatory requirements in a given variant.